1. Field of the Invention
The present invention relates to a novel peptide which comprises a sequence of amino acids reproducing more particularly the interaction site between sub-unit A and sub-unit B of cholera toxin.
2. Description of the Background
As is known [Cf. J. HOLMGREN, NATURE, 292, 30 July 1981, p. 413-417] the watery diarrhea characteristic of cholera which, if it is not arrested, leads to dehydration, to metabolic acidosis and death, is due to the cholera toxin secreted by Vibrio cholerae. The mechanism of action of cholera toxin is well known; it fixes itself to receptors which are present on the mucous cells and stimulates intestinal adenyl cyclase activity, which has the effect of increasing the cyclic A.M.P. level present in the cells of the small intestine; the latter causes diarrhea and fluid loss through severe water depletion and inhibition of the absorption of sodium chloride by the intestinal villosities and by the stimulation of the active secretion of chlorine by the cryptic cells.
It is also known that cholera toxin (CT) is a protein constituted of two polypeptide fragments: sub-unit B itself comprising five identical units, and sub-unit A [Cf. R. A. FINKELSTEIN, M. K. LARUE and J. J. LOSPALLUTO, INFECT. IMMUN., 6, 1972, pp 934]. It is accepted [Cf. J. HOLMGREN, Loc. quoted above] that the injurious action of the cholera toxin is manifested by the following facts: fixation of the sub-unit B of the toxin to the GM1 receptor of the membrane of the duodenal enterocyst, followed by an increase of the cyclic A.M.P. level due to an irreversible activation of the adenyl cyclase [Cf. D. M. GILL, ADV. CYCLIC NUCLEOTIDE RES., 8, 1977, pp. 85] which would lead one to think that the symptomatology of cholera arises from the introduction of the sub-unit A into the cell.
This sub-unit A has a hydrophobic character which is demonstrated when it is fractionated from the cholera toxin (CT), by reduction of the disulfide bridge, to give non-identical sub-units .alpha. and .gamma.. According to W. H. J. WARD, P. BRITTON and S. VAN HEYNINGEN, (BIOCHEM. J., 199, 1981, pp 457), the .alpha. fragment would not have hydrophobic regions and, on the other hand, the .gamma. fragment would have an amphiphilic character, the hydrophobic regions being maskable in the intact sub-unit A. Finally, it would be due to the conductive fragment .gamma., that the .alpha. fragment would penetrate through the membrane of the cell. This is why, it appeared interesting and useful to Applicants to seek in the structure of the sub-unit A, the amino acid sequences responsible for the biological activities of the cholera toxin.
In addition, the investigations of E. K. SPICER, W. H. KAVANAUGH, W. S. DALLAS, S. FALKOW, W. H. KONIGSGERG and D. E. SCHAFER, reported in PROC. NATL. ACAD. SCI. U.S.A., 78, January 1981, p. 50-54 and those of W. S. DALLAS and S. FALKOW (NATURE, 288, 4 Dec. 1980, p. 499-501) have demonstrated the existence of similarities of primary structure between the cholera toxin (CT) and the thermolabile enterotoxin of Escherichia coli (LT) responsible for infectious gastro-enterites: the cholera toxin CT and the thermolabile toxin LT of Escherichia coli are similar enterotoxins from the functional, structural and immunological point of view. They both cause elevation of the cyclic A.M.P. levels in the epithelial cells of the intestines by catalysing NAD-dependant ADP-ribosylation of the membranal proteins. They are both composed of two dissimilar sub-units, namely sub-unit A of enterotoxin which has an enzymatic activity and is the activator component of adenyl cyclase and the sub-unit B which recognizes the membranal components and fixes the holotoxin to the target cell, juxtaposing the sub-unit A with its substrates. It has been demonstrated that the membranal receptors of the sub-unit B of the cholera toxin and of the Escherichia coli toxin are similar but not identical, and that the monosialosylganglioside GM1 is the receptor of the CT and constitutes probably a portion of the receptor of the LT. It has been demonstrated [Cf. CL GYLES and D. A. BARNUM, J. INFECT. DIS., 120, p. 419-426 (1978)] that the LT and the CT are similar from the immunological point of view and that both their sub-units A and their sub-units B have common antigenic determinants [cf. J. D. CELMENTS and R. A. FINKELSTEIN, INFECT. IMMUN., 21, p. 1036-1039 (1978)]. The primary structure of the sub-units B of the LT and of the CT has been determined and has shown that they have a homologous sequence of amino acids. The studies of SPICER et al and DALLAS et al mentioned above, have shown that there exists also similarities of primary structure and of their immunological properties between the sub-units A of the CT and of the LT and although the sequences of amino acids of these two sub-units are only partially identified at this time, it is however known that according to the studies of DUFFY and LAI [BIOCHEM. BIOPHYS. RES. COMM., 91, 1979, pp. 1005] the sub-unit A of the CT has a molecular weight of 29500, of which 24000 for the .alpha. chain and 5500 for the .gamma. chain whose composition in amino acids is known, which comprises 46 amino acids 11 of which are common with the .gamma. chain of the LT.
By taking stand on the hypothesis according to which the site of interaction between the sub-unit A and the sub-unit B of the CT would be localized in the .gamma. chain and more particularly in the sequence corresponding to the amino acids bearing numbers 10 to 24 from the terminal N of the latter, and by taking stand on the functional, structural and immunological similarity between the LT and the CT, Applicants considered that if they arrived at synthesizing a polypeptide having this sequence 10-24, they could obtain a protective agent both against cholera and against infectious gastro-enterites.